The bacterium Haemophilus influenzae inhabits the human nasopharynx and conjunctiva, and can disseminate to cause pneumonia, otitis media, or meningitis. The current vaccine against the capsule of type b strains is not effective against clinically relevant strains that lack the capsule, termed nontypeable H. influenzae (NTHi). After infection by influenza A virus (IAV), NTHi is associated with secondary pneumonia, a major cause of mortality in IAV infections. The lipooligosaccharide (LOS) of NTHi contributes to virulence in models of bacteremia and otitis media yet its function in the lung is not well understood. We have identified roles in the lung for several LOS structural genes involved in evasion of the complement (C) pathway, which targets NTHi for destruction by phagocytes. We have also found metabolic pathways critical for NTHi survival in the lung and depletion of neutrophils alleviates NTHi's requirement for these pathways. Understanding NTHi pathogenesis in the lung has been limited, in part because normal mice rapidly clear the bacterium and do not develop persistent pneumonia with NTHi alone. However, our preliminary data demonstrate extensive multiplication and rapid lethality of NTHi in mice infected with IAV. Therefore, the IAV/NTHi co-infection model provides an opportunity to examine previously uncharacterized pathogenic mechanisms of clinically relevant NTHi strains in the context of pulmonary disease. We hypothesize that immune evasion and physiological adaptations by NTHi within the respiratory tract allow this bacterium to exploit weakened host defenses in predisposing conditions such as IAV co-infection, thereby potentiating disease. We propose to use IAV co-infection as a model system to characterize the molecular basis by which bacteria exploit host vulnerability in the lung. To perform the first comprehensive examination of the roles of the diverse colonization factors of H. influenzae in promoting pulmonary disease we will: Comprehensively compare bacterial adaptations and immune evasion factors required for growth and survival of NTHi in IAV co- infection versus those required in infection with NTHi alone; Characterize roles in immune evasion of NTHi LOS structures and antioxidant defenses that are differentially involved in IAV co-infection; and Determine mechanisms of NTHi pathogenesis in the context of altered host response and in IAV co-infection. Relevance: The proposed work will generate insight into the roles of bacterial genes of immune evasion and physiological adaptation required to survive anti-bacterial defenses modeled in vitro, and how these genes contribute to bacterial survival and colonization in lungs of both normal versus virally infected mice. The results will provide unique insight into strategies used by H. influenza and potentially other respiratory pathogens to resist defenses in the lung and exploit conditions in individuals with underlying disease. Moreover, understanding this bacterium's interactions with molecular components of its environment in normal and virally infected hosts will be valuable for the selection of appropriate targets for preventative or therapeutic strategies specifically designed to combat bacteria in viral co-infections and potentially other conditions that predispose individuals to bacterial lung infection.